Project Summary/Abstract Helminths, allergens, and certain protists all stimulate a type 2 immune response and contribute significantly to the global disease burden. Currently, more than 1 billion individuals worldwide are infected with helminths, and the rising incidence of allergic disease represents an emerging epidemic. The sensing and signaling events that initiate type 2 immunity remain poorly understood, but in the small intestine they require epithelial tuft cells. Tuft cells regulate a tuft-ILC2 circuit in which tuft cell-derived IL-25 activates group 2 innate lymphoid cells (ILC2s) in the underlying tissue. ILC2s secrete the canonical type 2 cytokines IL-5, -9, and -13, which collectively drive hallmarks of type 2 immunity, such as eosinophilia and tissue remodeling. IL-13 also promotes a feed-forward response by inducing tuft and goblet cell hyperplasia. The immune function of tuft cells requires a chemosensory pathway and recent studies identified the microbial metabolite succinate as an intestinal tuft cell ligand that is sufficient to activate the tuft-ILC2 circuit. Tuft cells therefore act as IL-25- secreting immune sentinels, but several lines of evidence support the central hypothesis of this proposal that additional tuft cell effector functions must exist: (1) Tuft cells express IL-25 constitutively, but the feed-forward tuft-ILC2 circuit is only activated in the presence of helminths or protists, suggesting additional activating signals; (2) the initiation of tuft cell hyperplasia after immune sensing has occurred suggests tuft cells contribute to the effector stages of type 2 immunity; and (3) helminth clearance is more delayed in tuft cell- deficient mice than in mice that lack only IL-25. The goal of this proposal is therefore to discover and characterize novel tuft cell effector functions in the small intestine. Using a combination of innovative in vitro assays and in vivo helminth infection of genetically modified mouse strains, we propose to test the regulation and function of tuft cell-derived IL-25, leukotriene C4, and acetylcholine. These studies should identify novel targets for therapeutic intervention in both helminth infection and allergic disease.